The main obstacle to improving survival, or indeed altering the management of prostate cancer (CaP) patients, is our failure to prevent the development of, or have adequate treatment for, androgen-independent CaP. In order to overcome this obstacle, we need to understand the molecular events that occur which allow CaP to become androgen independent (AI). In this proposal, we will investigate the molecular abnormalities occurring in AI CaP. We will explore the roles that the human androgen-receptor (hAR, or AR) and AR-related molecular alterations play in the progression of CaP. In our initial grant we developed four strategies that we used successfully to determine the role of the AR in CaP. We will now extend these strategies into large-scale studies to elucidate the mechanisms involved in various aspects of the development and progression of CaP. We hypothesize that CaP cells carrying multiple AR-related molecular alterations are more likely to progress rapidly to androgen independence. [unreadable] We will pursue three specific aims to test this hypothesis. Aim 1 is to determine the types and frequencies of AR-related molecular alterations in clinical prostate cancer specimens and ascertain their relationships to patient relapse and survival. In Aim 1, we will: 1) use a colorimetric yeast functional assay to detect structural alterations occurring in the full coding region of the AR, 2) employ both immunohistochemistry and/or flourescence in situ hybridization to determine the extent of AR amplification and overexpression, and determine the expression levels of AR coactivators. The goal of Aim 2 is to evaluate the effects of multiple AR-related molecular alterations on AR functionality using the quantitative yeast system and an AI-CaP cell model. The molecular alterations to be explored in Aim 2 are the effects of AR mutations, short CAG repeat lengths in exon 1 of the AR and the influence of AR-related cofactors on the hAR function. Aim 3 is to evaluate the effects of AR-related molecular alterations on the biology of CaP. We will focus on CaP tumorigenesis using an immortalized prostate epithelial cell line. We will learn whether transfecting altered ARs and AR-related cofactors into these cells will permit them to grow as tumors in nude mice. We will also explore gene expression profiles that reflect the conversion of these cells from a non-tumorigenic to a tumorigenic state. Our proposed studies should provide important new information for elucidating the functional biology of mutant ARs and their coactivators, and lead to identifying target molecules for development of potential therapies for advanced CaP. [unreadable] [unreadable]